Cultured microorganisms are the most common source of antibiotics and other medicinal agents. However, only a small percentage (less than 1%) of the viable bacteria in soil can be cultured on known nutrient media using current techniques such as petri dishes (Handelsman et al. 1998; Amann et al. 1995). The other 99% of uncultured/uncultivable microorganisms, with their genetic and biochemical diversity, may emerge as a major source of new natural chemical structures that may be useful for humans, for example as drugs.
The exploration of previously uncultured microorganisms for the discovery of new useful natural products is now being carried out in several laboratories. The main approach involves genomics techniques such as the approach designated metagenomics for the analysis of the collective genomes of the microorganisms in the soil community. According to this approach, DNA in large segments is cloned directly from soil into a culturable host and a sequence-based and functional genomic analysis is conducted on it. The intention is the isolation of new signals, new secondary metabolites that might have utility for humans, and the reconstruction of an entire genome of an uncultured organism.
Molecular microbial ecology represents a recent development in research methods. It consists of utilizing techniques of molecular biology to investigate the ecology of microorganisms, and offers new tools to facilitate the detection and identification of microorganisms in the environment.
Molecular microbial ecology allowed the development of tools to address a central dogma of microbial ecology: an inability to cultivate more than a small proportion (0.1-1%) of the bacteria that can be visualized by direct count procedures (Head et al., 1998). Thus, the identification of bacteria by molecular methods represents an indispensable addition to the traditional methods based on the analyses of morphological and physiological characteristics. Among these culture-independent new techniques, the technique based on direct sequencing seems to be the most effective. It consists in sequencing a specific region of the bacterial chromosome, namely the bacterial 16S rDNA region, and in comparing this sequence with known sequences stored in data banks. All microorganisms possess at least one copy of the genes coding for the ribosomal RNA (rRNA), which are indispensable in any cells for the biosynthesis of proteins. Within these genes, the 16S rDNA region is principally used for the determination of the genus and the species of bacteria. By using this approach, it could be determined in many environmental samples the predominance of many different uncultured species. It might be feasible that the yet uncultured types of bacteria might be grown under laboratory conditions if just the right nutrients are found (Amann et al. 1995; Felske et al. 1999).
Recently, Kaeberlein et al. (2002, and published US Patent Applications Nos. 2003/0059866 and 2003/0059867) disclosed a new method for isolating and growing uncultivable microorganisms in pure culture in a simulated natural environment using a diffusion chamber. Microorganisms were separated from intertidal marine sediment particles, serially diluted, mixed with warm agar made with seawater, and placed in the diffusion chamber. The membranes allowed exchange of chemicals between the chamber and the environment, but restricted movement of cells. After the first membrane was affixed to the base of the chamber, the agar with microorganisms was poured in, and the top was sealed with another membrane (See FIG. 1, Kaeberlein et al., 2002' and FIG. 1a, US Patent Application No. 2003/0059866). The diffusion chamber consists of a stainless steel washer (70 mm o.d., 33 mm i.d., 3 mm in thickness) sandwiched between two 0.03-μm pore-size polycarbonate membranes. The membranes were glued to the washer forming the inner space filled with test microorganisms in semi-solid agar. The sealed chambers were placed on the surface of the sediment collected from the tidal flat and kept in a marine aquarium. Colonies of representative marine microorganisms were isolated in pure culture. These isolates did not grow on artificial media alone but formed colonies in the presence of other microorganisms.
Zengler et al. (2002) disclose a universal method that provides access to the immense reservoir of untapped microbial diversity. They utilized a technique that combines encapsulation of cells in gel microdroplets (using the OneCell System technology) for massively parallel microbial cultivation under low nutrient flux conditions, followed by flow cytometry to detect microdroplets containing microbial microcolonies.
In summary, most microorganisms in the environment have been overlooked as yet due to their resistance to cultivation on artificial media. The cultured microorganisms represent only a small fraction of natural microbial communities and hence the microbial diversity in terms of species richness and species abundance is grossly underestimated. Our understanding of microbial diversity is not represented by the cultured fraction of the microbial community (Wintzingerode et al., 1997).